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024 Poster abstracts / Biochemical

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ffects of incorporation of �5 subunits into �4�2 nicotiniccetylcholine receptors on pharmacological functions of nico-inic ligands

enny Zhang ∗, Merouane Bencherif, Terry Hauser

Targacept, Inc., Winston-Salem, NC, USA

o elucidate their role in diseases and disorders such as addiction,ain and attention deficit/hyperactivity disorder, the heteromeric4�2 nicotinic acetylcholine receptors (NNRs) have been the focusf many studies. Recently, investigations have shown that otherembers of the NNR family, such as �5 expressed with �4�2 in

he brain, may play important roles in these diseases. Receptorunctions involving activation and desensitization are influencedy many factors including subunit composition which is likelyodified during the course of diseases. In the present study, we co-

ransfected concatenated �2–�4 dimers with single �4, �2 or �5onomers, to express low sensitivity (LS)-�4�2, high sensitivity

HS)-�4�2 or (�4�2)2�5 (�4�2�5) NNRs respectively in HEK293Fells. Subsequently the effect of �5 incorporation on the phar-acological properties of �4�2 agonists varenicline, cytisine, and

azetidine-A was investigated by a membrane potential method.or varenicline and cytisine, the activation profiles were very sim-lar on all three �4�2 NNRs. They partially activated LS-�4�2 withomparable potencies but had no detectable activities at HS-�4�2NRs. At activation of �4�2�5, similar potency and efficacy werebserved as compared to their respective responses at LS-�4�2NRs. Sazetidine-A, in contrast, was a potent full agonist at HS-4�2 with low activity at LS-�4�2. Its partial agonistic activityt �4�2�5 was similar to that at LS-�4�2. Interestingly, whenells were pretreated with these agonists and then challenged withcetylcholine (ACh), they all displayed similar desensitization effi-acy, to a complete degree, at the three �4�2 NNRs, again withomparable potencies for varenicline and cytisine. Additionally,nset of the desensitization was within minutes for all agonistsested and fastest for sazetidine-A. We also characterized thenfluence of �5 on sensitivities of �4�2 NNRs to the antagonistsihydro-beta-erythroidine (DH�E) and TC-5280. When co-appliedith ACh, DH�E and TC-5280 displayed IC50 values of 114 and

77 nM at HS-�4�2, 123 and 364 nM at LS-�4�2, and 85 and39 nM at �4�2�5, respectively. Under pre-treatment condition,C-5280 was more potent and selective to desensitize HS-�4�2nd �4�2�5 NNRs. In summary, for three agonists tested, incorpo-ation of �5 shifted their activation responses to LS-�4�2-like andheir desensitization responses to HS-�4�2-like. Understandinghe changes in pharmacological functions influenced by receptorubunit composition will help us to understand the physiological

ole of such receptors and to better design drugs that target theelevant receptor subtypes involved in diseases.

oi:10.1016/j.bcp.2011.07.005

acology 82 (2011) 1023–1047

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Comparison of binding and functional activity profiles for a setof nicotinic acetylcholine receptor ligands across multiple �6*expression systems

Scott R. Breining 1,∗, Christopher Hepler 2, Paul Whiteaker 3,Maryka Quik 4, Sharon R. Grady 5, Daniel Yohannes 1

1 Drug Discovery, Targacept, Inc., Winston-Salem, NC, USA2 Neurochemistry, Targacept, Inc., Winston-Salem, NC, USA3 Neurobiology, Barrow Neurological Institute, St. Joseph’s Hospitaland Medical Center, Phoenix, AZ, USA4 SRI International, Menlo Park, CA, USA5 Institute for Behavioral Genetics, University of Colorado, Boulder, CO,USA

It has been proposed that modulation of the nicotinic acetylcholinereceptor �6* (where the asterisk indicates additional subunits) sub-type may offer therapeutic opportunities in overcoming nicotineaddiction and in alleviating motoric dysfunction associated withParkinson’s disease. Progress in the identification of selective �6*ligands has previously been hampered by the lack of robust, phar-macologically relevant high-throughput assays. Until recently, theonly assay amenable to high throughput in vitro screening was the�6/4�4 chimera. Native �6* subtypes associated with dopamin-ergic activity are believed to contain an �6-�2 interface whichis missing in the �6/4�4 chimera, calling into question the rele-vance of this data for predicting in vivo activity in animal modelsof addiction and Parkinson’s disease. We have recently identifieda number of compounds with moderate to high affinity for �6*-containing receptors through screening of previously publishednicotinic ligands and Targacept proprietary database libraries ina variety of assay formats. These include novel �6�3�4�5 con-struct expressed in mammalian cells, rat native tissue from �6-richbrain areas, an �6/3�2�3 chimera expressed in mammalian cells,and autoradiography in rat striatum. The affinity and, consequently,selectivity versus other nicotinic receptor subtypes (�4�2, �3�4)varied greatly depending on the assay system. We present herethe in vitro profiles of these diverse ligands across multiple �6*affinity measures and preliminary structure–activity relationships.The relevance of �6* binding affinity to �6* function, as measuredby Calcium flux in the �6/3�2�3 chimera and the �-conotoxinMII-sensitive component of striatal dopamine release, will also bediscussed.

doi:10.1016/j.bcp.2011.07.006

1.5

Construction of cell line heterologously expressing the�6/3�2�3 nicotinic acetylcholine receptor (nAChR) subtype

P. Whiteaker 1,∗, L. Lucero 1, R.J. Lukas 1, C. Hepler 2, J.P. Strachan 2,S. Letchworth 2

1 Division of Neurobiology, Barrow Neurological Institute, Phoenix, AZ,USA2 Targacept, Inc., Winston-Salem, NC, USA

Natively-expressed �6* nAChRs are predominantly expressed incombination with �2 and �3 subunits (�6�2�3* nAChRs; aster-isk denotes other possible subunits). Heterologous expression offunctional �6* nAChRs in mammalian cell lines has historicallyproved difficult. In order to overcome this problem, we have applied

a combination of approaches to increase expression of functionalnAChRs, without using a gain-of-function mutation that could alterEC50 and IC50 values or change agonist efficacies. Native �6 sub-units were replaced with �6/3 chimeric subunits, which have